Atherosclerosis is the number one cause of mortality in Western societies. Therefore, finding new and more effective anti-atherogenic therapies is critical to saving many lives. PON1 is a serum enzyme that has been implicated in playing a protective role against development of atherosclerosis. The long-term objective of this research is to test the hypothesis that enhancement of PON1 catalytic activity by a selective activating agent will attenuate atherosclerosis in a mouse model of the disease. Success in this objective may provide proof-of-concept data for a novel therapeutic strategy. The specific goal of this research proposal is to test the hypothesis that aptamers can be discovered that enhance PON1 enzymatic activity. Specific aim 1 is to generate a mechanism-based activity probe (AP) that covalently modifies PON1 upon catalysis and thus labels active enzyme with biotin. Specific aim 2 is to isolate PON1 activating aptamers and specific aim 3 is to characterize the aptamers. Aptamers will be isolated that activate PON1 catalytic activity through a novel positive selection scheme involving exposure of purified PON1 to a random DNA library followed by a limiting concentration of AP. Thus, a competition for limiting AP will be established which will result in preferential biotin labeling of catalytically more active PON1 molecules activated by a bound aptamer. The bound DNA will be isolated by capturing biotinylated PON1 using streptavidin conjugated beads. This DNA will be amplified by PCR, converted to single-stranded DNA and the selection cycle repeated with a lower concentration of AP. Thus, aptamers that have enhancing activity when bound to PON1 will be preferentially isolated with each round of selection. In addition, the competition for probe will result in selection for aptamers with the greatest PON1 activation activity. The aptamers isolated by this scheme will be characterized for potency and efficacy in activating PON1 with and without reconstituted HDL particles with three different substrates representing the three substrate classes for PON1. These data will aid in preparing and prioritizing aptamers as tools for future in vitro and in vivo experiments to determine whether activating PON1 catalytic activity will enhance the antiatherogenic activities of PON1. Atherosclerosis is the number one cause of mortality in Western societies. This research proposal focuses on generating research tools to evaluate a novel therapeutic strategy to prevent atherosclerosis.